Method to Produce Chemical Pattern In Micro-Fluidic Structure

ABSTRACT

A flow cell includes: a first substrate; a second substrate; a first resin layer disposed over an inner surface of the first substrate; a second resin layer disposed over an inner surface of the second substrate; a first plurality of biological capture sites located at the first resin layer; a second plurality of biological capture sites located at the second resin layer; and a polymer layer interposed between the first resin layer and the second resin layer, such that the first substrate is attached to the second substrate via at least the first resin layer, the polymer layer, and the second resin layer, wherein the polymer layer defines a plurality of microfluidic channels that extend through polymer layer.

This application is a divisional of U.S. patent application Ser. No.15,167,764, filed on May 27, 2016, and entitled “Method to ProduceChemical Pattern In Micro-Fluidic Structure” which is a divisionalapplication of U.S. patent application Ser. No. 14/040,303 filed on Sep.27, 2013, now U.S. Pat. No. 9,352,315 issued on May 31, 2016, entitled“A Method to Produce Chemical Pattern in Micro-Fluidic Structure,” isincorporated herein by reference.

FIELD

This disclosure relates to biosensors and methods for formingbiosensors. Particularly, this disclosure relates to micro-fluidicdevices and methods for forming them.

BACKGROUND

Biosensors are devices for sensing and detecting biomolecules andoperate on the basis of electronic, electrochemical, optical, and/ormechanical detection principles. Biosensors can sense charges, photons,and mechanical properties of bio-entities or biomolecules, or throughmolecular tags. The detection can be performed by detecting thebio-entities or biomolecules themselves, or through interaction andreaction between specified reactants and bio-entities/biomolecules.Biosensors continue to be miniaturized to reduce sample size whileincreasing sensitivity and information content.

A flow cell is a type of biosensor that includes micro-fluidicstructures that allows external detection of its contents through atransparent window, for example, with microscopes, spectroscopes, orrefractometers. The flow cell includes many capture sites on whichbiochemical reactions occur. The capture sites may be patterned orunpatterned (randomly distributed) on or in one or several microfluidicchannels. Flow cells may be used to analyze biomolecules, conductreactions, and irradiate samples. For example, flow cells may be used tofor deoxyribonucleic acid (DNA) sequencing using fluorescent dyes foroptical sensing.

Optical sensing techniques continue to improve, primarily from usingbetter cameras with more pixels and better sensitivity to obtain moreinformation from a flow cell. To benefit from the improved opticalsensing techniques, the capture sites in flow cells are furtherminiaturized using semiconductor processing techniques. Challenges infabrication of the flow cells using semiconductor processes arise, forexample, due to compatibility issues between the semiconductorfabrication processes, the biological applications, and restrictionsand/or limits on the semiconductor fabrication processes.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure are best understood from the followingdetailed description when read with the accompanying figures. It isemphasized that, in accordance with the standard practice in theindustry, various features are not drawn to scale. In fact, thedimensions of the various features may be arbitrarily increased orreduced for clarity of discussion.

FIGS. 1A and 1B are a cross section view of a flow cell in accordancewith various embodiments of the present disclosure;

FIG. 1C is a top view of a flow cell in accordance with variousembodiments of the present disclosure;

FIGS. 2A and 2B are flow charts of various embodiments of methods offabricating a flow cell according to one or more aspects of the presentdisclosure;

FIGS. 3A-3L are cross-sectional views of a flow cell in accordance withvarious embodiments according to methods of FIGS. 2A and 2B of thepresent disclosure;

FIG. 4 is a flow chart of some embodiments of methods of fabricating aflow cell according to one or more aspects of the present disclosure;

FIGS. 5A-5E are cross-sectional views of a flow cell in accordance withvarious embodiments according to methods of FIG. 4 of the presentdisclosure; and

FIGS. 6A-6J cross-sectional views of a flow cell in accordance with someembodiments of the present disclosure.

DETAILED DESCRIPTION

It is to be understood that the following disclosure provides manydifferent embodiments, or examples, for implementing different featuresof the invention. Specific examples of components and arrangements aredescribed below to simplify the present disclosure. These are, ofcourse, merely examples and are not intended to be limiting. Moreover,the formation of a first feature over or on a second feature in thedescription that follows may include embodiments in which the first andsecond features are formed in direct contact, and may also includeembodiments in which additional features may be formed interposing thefirst and second features, such that the first and second features maynot be in direct contact. Further still, references to relative termssuch as “top”, “front”, “bottom”, and “back” are used to provide arelative relationship between elements and are not intended to imply anyabsolute direction. Various features may be arbitrarily drawn indifferent scales for simplicity and clarity.

A flow cell involves various bioreceptors which react with variousbiological material of interest in one or more patterned capture sites.One such reaction is the polymerase chain reaction (PCR) that canmultiply by orders of magnitude the number of molecular strands at asite. Various approaches are used to differentiate among differentreactants and reactions for detection. One common approach is to tag areaction with a fluorescent or phosphorescent label that emits adetectible photon that can be photographed externally. Different tagscan be made to emit a different fluorescence, phosphorescence, orotherwise detectible and differentiable radiation. Nature of thedetection would be determined based on the type of signal transduced.The signal transduced may be photons, for example, where a differentwavelength of light is generated for different biological materials orreactions. In the example of deoxyribonucleic acid (DNA) sequencing, adifferent color may be emitted for a different DNA-base (i.e., A, G, C,and T).

The bioreceptors are located within various microfluidic channels thatdeliver biological material to the sensors. The microfluidic channelsmay be a part of a microfluidic system that includes pumps, valves, andvarious measurement devices such as flow meters, pressure transducers,and temperature sensors. Combinations of fluid processing and sensingmay be advantageously integrated.

Semiconductor processing often involves baking, curing, and exposingvarious surfaces to plasma energy and radiation energy. At hightemperatures (i.e., above about 100 degrees Celsius, or over 150 degreeCelsius) and/or high energies, these processes would damage or destroyorganic bioreceptors and capture sites, which usually are delicatebio-molecules or very thin layers of surface chemistry. According tovarious embodiments. the surface modification chemistry of capture sitesincludes an adhesive promoter layer, for example, hexamethyldisilazane(HMDS) or 3-aminopropyl triethoxysilane (APTES), and a bio-medium layer,for example agar, or hydrogel. In some embodiments, the adhesivepromoter layer and the bio-medium layer may be mixed and applied to thesurface together. The adhesive promoter, usually a form of silane,attaches to silicon-containing surfaces. The bio-medium layer attachesto the adhesive promoter. Without the adhesive promoter, a bio-mediummay detach from a silicon-containing surface.

Thus, the bio-functionalization of surfaces on which bio-molecules areattached, are often performed after all the semiconductor processes arecompleted to avoid being exposed to the high temperature processes. Insome designs, the microfluidic channels are formed directly on asubstrate, usually a transparent substrate such as quartz, or an opaquesubstrate such as a silicon wafer, along with capture sites. At leastone side of the flow cell is transparent to allow optical sensing ofemitted radiation. By use of the term “transparent,” it is not intendedthat the disclosure be limited to substrates that are 100% lighttransmissive. Rather, transparent is used herein consistently withcommon usage by those in the field to indicate transmissivity sufficientto obtain the desired functionality of the resulting device.

In other designs, the microfluidic channels are formed on a microfluidicsubstrate that is subsequently bonded to another substrate having thecapture sites. In the case where the microfluidic channel is in one ofthe substrates, the microfluidic channel formation, usually etching atrench or via into the substrate, can be difficult to manufacture on atransparent substrate. To avoid damage, when a high temperature bondingprocess is used, the temperature-sensitive materials are deposited onthe interior walls of the microfluidic channels after the bondingprocess and the microfluidic channels are enclosed. A high concentrationof material is flowed through each flow cell through the microfluidicchannel surfaces having some affinity for the material. However, thedensity and thickness of material that attaches to the surfaces are hardto control, and the process is slow and wasteful of thetemperature-sensitive material. In some cases, the material densityvaries throughout the flow cell or a batch of flow cells (not uniform)as the concentrations in the reagents change from inlet to outlet. Therandom, non-aligned locations and non-uniform concentrations complicateresolution of detectible activities at different sites using imageprocessing algorithms. The locations may overlap each other and are hardto resolve. The randomness also makes it difficult to correlate betweendifferent flow cells because each would have different mapping ofcapture sites.

The various embodiments of the present disclosure contemplate awafer-level process and a flow cell that addresses many of these issuesby avoiding high temperature processing and/or allowing the use of densepatterns of capture sites without traditional photolithography on atransparent substrate. The bioreceptors, for example, DNA primers, areattached to the capture sites after the flow cell is fabricated. Thesite locations are patterned with an adhesive promoter layer and abio-medium to form the capture sites. The density issue and the random,non-aligned location issue are addressed by forcing the bioreceptors toattach only at the patterned capture sites. The material selected forcapture sites allows certain semiconductor processes to be used infabricating the flow cell that otherwise cannot be used, for example,with processes that use a low temperature baking and curing. The variousmethod embodiments of the present disclosure may be performed in asemiconductor fabrication facility. In more detail, the processing ofmaterials on transparent substrates such as quartz and glass and etchingof transparent substrates are often incompatible with some stages of theCMOS process because, for example, the glass processing can introduceparticles that are considered contaminants for other CMOS processes.Some embodiments of the present disclosure involve no glass processingor minimal processing of glass when it is used as a transparentsubstrate.

In certain embodiments, the flow cell of the present disclosure isformed by combining two substrates, at least one of which istransparent. FIGS. 1A-1C are cross-sectional views and a top view of aflow cell 100 in accordance with some embodiments of the presentdisclosure. FIG. 1A is a cross-section of the flow cell 100 fromsectional line A-A′ of FIG. 1C. The view of FIG. 1B is cut fromsectional line B-B′ of FIG. 1C. Flow cell 100 includes a first substrate101 bonded to a transparent substrate 103. The first substrate 101 has amicrofluidic inlet 107/109 and a microfluidic outlet 109/107.Microfluidic channel patterns are shown as channels 111, 113, and 115.The various channels 111, 113, and 115 are connected to each other viavarious pathways and may be different sizes depending on the design ofthe flow cell. The channels include various capture sites 117 on bottomof the channels close to the transparent substrate 103 or top of thechannels on the first substrate 101, or both. FIGS. 1A and 1B show thecapture sites 117 on both top and bottom of the channels 111 and 113.Capture sites 117 are formed on the first substrate 101 and thetransparent substrate 103. The capture sites 117 may have differentdensities in different channels. In some embodiments, the capture sites117 on the first substrate 101 and the transparent substrate 103 arealigned in a top view. In other embodiments, the capture sites 117 onthe first substrate 101 and the transparent substrate 103 are offset ina top view such that an optical detector sees only one capture site 117per location.

The capture sites have certain chemistries that allow some material tobind to it. According to various embodiments, a bio-medium on whichvarious bio-materials can bind, including agar or polyethylene glycol(PEG) hydrogel, is disposed on the capture sites. The bio-medium isconnected to the capture site on the substrate through an adhesionpromotion layer, which may be 3-aminopropyl triethoxysilane (APTES), orhexamethyldisilazane (HMDS).

A bonding polymer layer is disposed between the first substrate 101 andthe transparent substrate 103. The bonding polymer layer 119 defines themicrofluidic channels 111, 113, and 115 and seals the adjacentmicrofluidic channels from each other. The bonding polymer layer 119adheres to both the first substrate 101 and the transparent substrate103 and also seals the flow cell 100 along the perimeter such that theonly access to the microfluidic channels 111, 113, and 115 is throughthe inlet/outlet 107/109.

FIG. 2A is a flow chart of some embodiments of methods 200 offabricating a flow cell device according to one or more aspects of thepresent disclosure. FIGS. 3A to 3L are cross-sectional views ofpartially fabricated flow cell devices constructed according to one ormore steps of the method 200 of FIG. 2A.

In operation 202 of FIG. 2, through holes are laser drilled in a firstsubstrate or a second substrate. Other techniques of formingthrough-holes include various etching techniques and waterjet drilling.Laser drilling of cylindrical holes generally occurs through melting andvaporization (also referred to as “ablation”) of the substrate materialthrough absorption of energy from a focused laser beam. Depending on thedirection of the laser energy, the laser drilled through-holes can havean inverse trapezoidal shape in a cross section. In some embodiments,the through-holes are formed by microblasting, or ultrasonic drilling.Microblasting removes material by driving a high velocity fluid streamof air or inert gases including fine abrasive particles, usually about0.001 in (0.025 mm) in diameter. Ultrasonic drilling involves using highfrequency vibrations to hammer a bit through materials. Depending on theprocess used for forming the through-holes, by products need to beremoved from the substrate, by etching or cleaning.

At least two through-holes are formed for every flow cell—an inlet andan outlet. More than two through-holes may be used for different inletfluids or if the flow cell performs separation of the analyte and morethan one outlet is used. The through holes may be formed on separatesubstrates or the same substrate. Two through holes may be formed on thesame substrate, either the first substrate or the second substrate. FIG.3A is a cross sectional diagram of a substrate 301 before thethrough-holes is formed. FIG. 3B is a cross sectional diagram of asubstrate 301 having a through-holes 303 therein.

In operations 204 to 208, a number of first capture sites is formed on afirst substrate. In operation 204, a nanoimprint lithography (NIL) stampstamps a patterned adhesive promoter on a first substrate. In an NILprocess, a sample liquid, also called ink, is transferred to a planartarget surface in a stamping motion. The stamp may be a siliconematerial such as polydimethylsiloxane (PDMS), a silicon material, or asilicon oxide material. The island features are formed by molding,photolithographic patterning, or ion beam patterning. Depending on theprocess used, the island features may be small with a small pitch. Forexample, the island features may be tens of angstroms across with apitch in the same range. In some examples, each island may have a widthand a length of about 10 to 50 angstroms. In other examples, each islandmay have a width and a length of about 50 to 200 angstroms. The islandsize is determined from the use of the flow cell. For DNA sequencingwhere smaller clusters are detectible by the optical sensinginstruments, the island features may be about 20 angstroms. If the flowcell is used to work with cells or large biomolecules, larger islandsmay be used.

The island features contact the liquid sample in a reservoir, much likea stamp first contacts an ink well. Some liquid sample is retained onthe island surface by the island feature. The stamp is then pressed ontoa surface of the substrate to transfer at least a portion of the liquidsample from the island surface to the substrate surface. FIG. 3C showsthe first substrate 301 and a NIL stamp 305 over the first substrate301. The first substrate 301 may be quartz, silicon, sapphire, siliconcarbide, or transparent or non-transparent substrates that do not reactwith the analyte. The NIL stamp 305 includes many island features 307having an adhesive promoter 309 on its surface. The adhesive promoter isa liquid chemical that bonds to the substrate 301, for example, asilane-based or thio-based molecule, and can bond to a bio-medium toanchor the bio-medium to the substrate. In some embodiments, theadhesion promoter is HMDS or APTES.

The NIL stamp 305 makes contact with the first substrate 301 and islifted as shown in FIG. 3D. A portion of the adhesive promoter 309 isleft behind and forms an adhesive promoter imprint on the firstsubstrate 301. The adhesive promoter imprint may be a monolayer. Thepatterned adhesive promoter 309 on the first substrate has the samepattern as the island features 307 on the NIL stamp 305. The adhesivepromoter transfer may not be complete, some adhesive promoter materialmay be left on the island features 307. Further, while adhesive promoter309 is inked only on the island features 307 in FIG. 3C, the adhesivepromoter 309 may be present in the valley areas of the NIL stamp 305between the island features 307. However, because only the islandfeature surface contacts the first substrate 301, the additionaladhesive promoter 309 between the island features does not affect thepattern imprinted.

In operation 206 of FIG. 2A, an NIL stamp stamps a bio-medium on thepatterned adhesive promoter on the first substrate. The adhesivepromoter and the bio-medium form a capture site. The patterned adhesivepromoter and the bio-medium on the patterned adhesive promoter form acapture site pattern. The NIL stamp used to stamp the bio-medium may bethe same NIL stamp used to stamp the adhesive promoter or a differentNIL stamp having the same island feature pattern. FIG. 3E shows thefirst substrate 301 and a NIL stamp 315 over the first substrate 301.The NIL stamp 315 includes many island features 307 with bio-mediummaterial 311 on the surface. The bio-medium material is a support mediumthat can bind to primers for performing reactions in the flow cell. Thebio-medium may be a natural or a synthetic material. In someembodiments, the bio-medium material is agar or a hydrogel, for example,polyethylene glycol (PEG) hydrogel.

The NIL stamp 315 makes contact with the first substrate 301 and islifted as shown in FIG. 3F. A portion of the bio-medium 311 is leftbehind on the adhesive promoter 309 on the first substrate 301. Becausethe bio-medium 311 is stamped directly on the adhesive promoter pattern309, the NIL stamp 315 is aligned carefully with the adhesive promoterpattern 309. The bio-medium 311 stamping may be performed more than onceto increase the thickness of the bio-medium 311 layer. The patternedbio-medium 311 on the first substrate has the same pattern as the islandfeatures 317 on the NIL stamp 315. In some embodiments, the NIL stamp305 of FIG. 3C and the NIL stamp 315 of FIG. 3E formed of differentmaterials having the same island feature pattern. The bio-mediumtransfer may not be complete, some bio-medium material may be left onthe island features 317. Further, while bio-medium 311 is inked only onthe island features 317 in FIG. 3E, the bio-medium 311 may be present inthe valley areas of the NIL stamp 315 between the island features 317.However, because only the island feature surface contacts the firstsubstrate 301, the additional bio-medium 311 between the island featuresdoes not affect the pattern imprinted.

Depending on the particular materials used for the adhesive promoter andfor the bio-medium, the substrate may be baked at a relatively lowtemperature to covalently bond the bio-medium and the adhesive promoter.In some embodiments, the covalent bonds are formed at a temperature of40 degrees Celsius to 200 degrees Celsius. In some embodiments, thematerials are irradiated to promote the covalent bonding using UV/Vis/IRlight for curing. A capture site includes an adhesive promoter layer anda bio-medium layer bonded over the adhesive promoter layer.

Referring back to FIG. 2A, in operation 210, a patterned polymer layeris formed on the first substrate or a second substrate. The patternedpolymer layer may be formed before or after the capture sites areformed. In some embodiments, the polymer layer is formed by using aphotoresist (PR) dry film. A PR dry film is applied to a substrate,either the first substrate or the second substrate. The PR dry film isthen exposed to a patterned light and developed to remove an unexposedportion of the PR dry film. In some embodiments, the exposed portion isremoved, depending on the type of PR dry film. The developer used todissolve the PR dry film is selected to be bio-compatible. The PR filmhas a low solvent content, so it would harden with a low temperaturebake at less than 100 degrees Celsius, for example, at about 90 degreesCelsius. At low temperatures, already formed capture sites would not beharmed. The patterned polymer layer becomes the walls of themicrofluidic channels as well as sealing the flow cell. FIGS. 3G and 3Hare cross sectional diagrams of a substrate 351 before and after formingthe patterned polymer layer 353. The patterned polymer layer 353 has athickness between about 10 microns to hundreds of microns. For example,the patterned polymer layer 353 may be about 100 microns.

Referring back to FIG. 2A, operations 212 to 216 on the second substrate(FIGS. 3I to 3L) mirror operations 204 to 208 on the first substrate(FIGS. 3C to 3F), with a different NIL stamp shape. Operations 212 to216 are used when capture sites are formed on the second substrate. Whena patterned polymer layer is formed before the capture sites, the NILstamp is shaped appropriately to be inserted between the polymerpatterns. According to some embodiments, the polymer layer pattern isused to align the NIL stamp. Referring to FIG. 3I, the NIL stamp 355having a channel base 356, island features 357, and an adhesive promoter359 is inserted between polymer pattern 353 to stamp an adhesivepromoter 359 on the second substrate 351. In some embodiments, thechannel base 356 has an angled side or two angled sides so as to form atrapezoidal shape to align the NIL stamp 355 to the microfluidic channelduring stamping. In FIG. 3J, the NIL stamp 355 is lifted from the secondsubstrate 351, leaving a patterned adhesive promoter 359 on the secondsubstrate 351. In FIG. 3K, an NIL stamp 365 stamps a bio-medium 361 onthe adhesive promoter 359. In FIG. 3L, the NIL stamp 365 is lifted fromthe second substrate 351, leaving bio-medium 361 behind. Operation 216is the same as operation 208 and may be used if the materials in thecapture site are bonded by a soft baking operation. In the embodimentsillustrated, a patterned polymer layer is formed on both the first andthe second substrate. In other embodiments, a patterned polymer layercould be formed on only one of the first or the second substrate, andthen bonded to the other of the second or first substrate.

Referring back to FIG. 2A, in operation 218, the first substrate and thesecond substrate are bonded via the patterned polymer layer on the firstsubstrate or the second substrate. In some embodiments, the patternedpolymer layers are formed on both the first substrate and the secondsubstrate in a non-overlapping or an overlapping matter. The overlappingembodiment may be used to increase the height of microfluidic channels.FIG. 2B is a process flow diagram showing operation 218 in more detail.In operation 220, the first substrate and the second substrate arealigned. The alignment may be performed by alignment marks on the firstsubstrate and the second substrate. If used, the alignment marks aredisposed in areas on the substrates outside of the flow cells. Thealignment marks may be disposed in scribe areas that will besubsequently removed when the bonded substrates are diced. The alignmentmarks may also be a part of the flow cells. For example, the alignmentmay be performed using the patterned polymer layer as the alignmentmarks.

In operation 222, the first substrate and the second substrate aremounted. In some embodiments, a transparent substrate is mounted on anon-transparent substrate when the alignment sensor can use features onthe non-transparent substrate detectible through the transparentsubstrate. After the substrates are mounted, in operation 224, the firstsubstrate and the second substrate are baked at a temperature less than100 degrees Celsius, with or without a downward pressure during thebaking. In addition or instead of baking, an ultraviolet (UV) light maybe applied to facilitate bonding of the polymer layer to a substrate.Once bonded, the patterned polymer layer forms a seal against thesubstrate bonded. The bonding operation may include one or many ofbaking, mechanical pressure application, and UV light. In certainembodiments, the UV light may be patterned so its application isdirected toward the areas of the polymer layer and not directed towardcapture sites. The photomask used for forming the patterned polymerlayer may be used to pattern the UV light.

After the bonding operation, any backside tape or handling apparatus isremoved from the bonded substrates. In one embodiment, an UV tape isapplied to one of the substrates to facilitate handling. The UV lightthat bonds the substrate and the polymer layer can also degrade theadhesive on the tape to render it easily removable. After the bondingoperation, the substrates may be diced or singulated to form individualflow cells as shown in FIGS. 1A to 1C. The tape removal may occur beforeor after the singulation. The flow cell thus formed has patternedcapture sites having small dimensions and high density to enable moreinformation to be captured during the biological reaction, such as DNAsequencing. The time to sequence a target is reduced. The NIL stampingprocess allows the patterned polymer layer to be formed on a substratebefore the capture sites are formed, reducing the likelihood thatdeveloper chemicals or other parts of the photolithography process usedfor the PR dry film affect the chemicals on the capture sites. Further apotentially damaging operation of using chemical mechanical polishing(CMP) to form capture sites in etched divots is avoided. This methodalso allows the use of transparent substrates in the semiconductor fabwithout contamination issues from etching of glass or quartz substrates.

The present disclosure also pertains to alternate methods of formingcapture sites on a substrate using an NIL process. FIG. 4 is a processflow of the method 400 to form capture sites. Cross sectional diagramsin FIGS. 5A to 5E correspond to various operations in method 400. Inoperation 402, a substrate is provided having a base substrate, a softbottom layer on the base substrate, and a coated top layer.

FIG. 5A is a cross section of substrate 500 including the base substrate501, the soft bottom layer 503 and a coated top layer 505. The basesubstrate 501 may be a transparent substrate or a non-transparentsubstrate. The soft bottom layer 503 is softer than an NIL stamp andallows the NIL stamp to deform the soft bottom layer 503. The softbottom layer 503 may be a thermal plastic resin or a metal layer. Themetal layer may include one or more of aluminum, copper, titanium, andan alloy of these. The soft bottom layer 503 has a thickness betweenabout 3 microns to about 10 microns. The soft bottom layer 503 may bedeposited on the substrate 501 using semiconductor deposition processesincluding physical vapor deposition (PVD), chemical vapor deposition(CVD), or spin-on processes. The coated top layer 505 is a silicon oxidehaving a thickness between about 1000 angstroms to about 5000 angstroms.The top coated layer 505 may be a spin-on glass (SOG) deposited usingspin-on processes. The SOG may be partially cured or fully cured and isthin and soft to allow an NIL stamp to punch through.

In operation 404 of FIG. 4, a surface chemical pattern is formed bystamping an NIL stamp through the coated top layer and stopping beneathan interface of the soft bottom layer and the coated top layer. FIG. 5Bincludes an NIL stamp 507. In the process of tamping. The NIL stamp 507punches through the coated top layer 505, forms an indentation in thesoft bottom layer 503, and stops without reaching the base substrate501. The NIL stamp 507 may be a silicon material, a silicon oxidematerial, a sapphire, or other hard material that can be patterned toform punch features 509. The punch features 509 are formed byphotolithographic patterning, or ion beam patterning. Depending on theprocess used, the punch features 509 may be small with a small pitch.For example, the punch features 509 may be tens of angstroms across witha pitch in the same range. In some examples, each punch feature 509 mayhave a width and a length of about 10 to 50 angstroms. In otherexamples, each punch feature 509 may have a width and a length of about50 to 200 angstroms. The punch feature pitch size is determined from theuse of the flow cell. For DNA sequencing where smaller clusters aredetectible by the optical sensing instruments, the punch feature pitchmay be about 40 angstroms. If the flow cell is used to work with cellsor large biomolecules, larger pitch may be used. The punch features 509have thickness determined by the thicknesses of the coated top layer 505and the soft bottom layer 503. Because the stamping is designed to stopbefore reaching the base substrate 501, the punch feature thickness isless than the sum of the coated top layer 505 and the soft bottom layer503.

The punch features 509 surfaces may be treated to increase its hardnessand to ensure that it does not bond during the stamping operation to thematerial on substrate 500. For example, an additional baking operationat about 200-460 degrees Celsius or UV curing may be used to hardenpunch features. The substrate 500 may be cleaned after the stampingoperation to ensure that no silicon oxide material remains at the bottomof the openings 511. The NIL stamp 507 is inspected and replaced ifnecessary to ensure mechanical integrity during the stamping operation.

The NIL stamp 507 is lifted as shown in FIG. 5C, leaving openings 511 inthe substrate 500 in the coated top layer 505 and soft bottom layer 503.The remaining portions of the coated top layer 505 are the surfacechemical pattern. Capture sites are formed on the surface chemicalpattern in subsequent operations. Therefore, the punch feature size doesnot define the size of the capture sites; rather, the spacing betweenthe punch feature size defines the capture site size.

Referring to FIG. 4, in operation 406 the substrate is exposed to anadhesive promoter that reacts and adheres only to the coated top layer.According to various embodiments, the soft bottom layer does not form abond with the adhesive promoter. In some embodiments, the soft bottomlayer is selected to be non-wetting with respect to the adhesivepromoter. The substrate may be exposed to the adhesive promoter througha spin and rinse processes. The adhesive promoter selectively adheres tosilicon-containing surfaces, especially the patterned coated top layerof silicon oxide. The soft-bottom layer covers and protects theunderlying base substrate from the adhesive promoter. Next, in operation408, the substrate is exposed to a bio-medium that attaches only to theadhesive promoter. Each patterned feature of the coated top layerbecomes a capture site having an adhesive promoter layer and abio-medium thereon.

FIG. 5D is a cross sectional diagram of substrate 500 after the adhesivepromoter 513 is attached to the patterned coated top layer. As shown,the adhesive promoter 513 is attached to the top and sides of thepatterned coated top layer, which is a silicon oxide island over anoutcrop of the soft bottom layer. The capture site is three-dimensional,as a box or a cylinder. In some embodiments, the capture site has a topsurface and four sidewalls. In other embodiments, the capture site has atop surface and a circumferential sidewall. The three-dimensional shapeallows more surface area to be used as capture site as compared to atwo-dimensional capture site having only a top surface.

In operation 410, the substrate is soft baked to covalently bond thebio-medium and the patterned adhesive promoter. Operation 410 is thesame as operation 208 of FIG. 2A. After operation 410 the capture sitesare formed on the substrate. The method 400 of FIG. 4 may be used toprepare capture sites on one or more substrates. FIG. 5E is a crosssection diagram of a flow cell 550. The flow cell 550 includes two basesubstrates 551 and 553, both with capture sites 555. The base substrate551, soft bottom layer 561, and capture sites 555 is the firstsubstrate. The base substrate 553, soft bottom layer 561, and capturesites 555 is the second substrate. Each capture site 555 includes asilicon oxide island formed from the coated top layer over an outcrop ofthe soft bottom layer 561 formed from the stamping operation.

The base substrate 553 includes a through hole 557, which may be formedafter the soft bottom layer and optionally the coated top layer isdeposited. Although only one through hole 557 is illustrated,embodiments are contemplated wherein two or more through holes areformed. A patterned polymer layer 559 is disposed between the firstsubstrate and the second substrate and defines microfluidic channelwalls. The polymer bonding process of operation 218 of FIGS. 2A and 2Bis used to attach the patterned polymer layer. While FIG. 5E shows thepatterned polymer layer disposed between two soft bottom layers 561,other embodiments may involve the patterned polymer layer directlycontacting the base substrates 551 and 553.

In some embodiments, capture sites are formed after the substrates arebonded. After the stamping operation 404 of FIG. 4, the substrates arebonded using the patterned polymer layer. Then the capture sites arecompleted by flowing the adhesive promoter layer and/or the bio-medium.Because the exposed coated top layer bonds with the adhesive promotermaterial selectively, the adhesive promoter may be flowed through themicrofluidic channel, rinsed, and then the bio-medium material may beflowed through the microfluidic channel and rinsed. This alternateembodiment has an advantage of not exposing the chemicals of the capturesites to any elevated temperature; however, the thicknesses of differentlayers on the capture site would be difficult to control.

According to some embodiments, a flow cell may include recessed capturesites as shown in FIGS. 6A to 6J. In FIG. 6A, a transparent substrate601 and a handling apparatus 603 is provided. The handling apparatus(opaque layer) may be a tape/polymer or deposited multi-layer films byPVD/CVD or another substrate bonded to the transparent substrate 601. InFIG. 6B, a number of recesses 605 are formed on the substrate. In someexamples, the recesses 605 are formed by patterning and etching thetransparent substrate 601. In FIG. 6C, capture sites 607 are formed inthe recesses. In some embodiments, the capture sites 607 are formed bysequential NIL stamping of an adhesive promoter and a bio-medium. Inother embodiments, the capture sites 607 are formed by a combination NILstamping of a mixture of adhesive promoter and bio-medium. In stillother embodiments, the capture sites 607 are formed by sequentialdeposition of an adhesive promoter and a bio-medium and a CMP to removeportions not in the recesses. In FIG. 6D, a patterned polymer layer 609is formed on the transparent substrate 601 according to previouslydescribed operation 210 of FIG. 2A.

In FIG. 6E, a second substrate 611 is provided. The second substrate 611may be transparent or not. In FIG. 6F, recesses 613 are formed in thesecond substrate 611 in a similar process as forming recesses 605 in thefirst substrate 601. In FIG. 6G, one or more through holes 615 areformed in the second substrate 611, in a similar process as described inassociation with operation 202 of FIG. 2A. In FIG. 6H, capture sites 617are formed in the second substrate 611 in a similar process as formingcapture sites 607 in the first substrate.

In FIG. 6I, the two substrates from FIG. 6D and FIG. 6H are bonded usinga polymer bonding process as described in association with operation 218of FIG. 2A. In FIG. 6J, the handling apparatus 603 is removed from theflow cell of FIG. 6I.

According to various embodiments, the capture sites in a flow cell mayhave a relatively flat surface (FIGS. 1A to 1C), a protrudingthree-dimensional (3-D) structure (FIG. 5E), or a recessed structure(FIG. 6J). Each capture site configuration may be manufactured using aNIL stamping process as described. In one flow cell, the capture siteson one side may have a different configuration from the other side. Forexample, in some embodiments, one side of the flow cell may have aprotruding 3-D capture site while the opposite side may have recessedcapture sites. Capture site design consideration includes the intendedflow rates in the microfluidic channel, size of the biological sample,the nature of intended reaction in the flow cell, the importance ofmaximizing surface area or separating clusters, manufacturing cost,among others.

One aspect of the present disclosure pertains to a method ofmanufacturing a flow cell that includes forming stamping a patternedadhesive promoter on a first substrate using nanoimprint lithography,stamping a bio-medium on the patterned adhesive promoter on the firstsubstrate using nanoimprint lithography to form a first capture sitepattern, forming a patterned polymer layer on the first substrate or asecond substrate, and polymer bonding the first substrate to the secondsubstrate. The first substrate or the second substrate or bothsubstrates are transparent. The method may also include soft baking thefirst substrate after stamping a bio-medium to covalently bond thebio-medium and the patterned adhesive promoter. In some embodiments, themethod also includes stamping a patterned adhesive promoter on thesecond substrate using nanoimprint lithography and stamping a bio-mediumon the patterned adhesive promoter on the second substrate usingnanoimprint lithography to form a second capture site pattern. The firstcapture site pattern and the second capture site pattern may be alignedin a top view. In some embodiments, the method also includes laserdrilling one or more through holes in the second substrate. The polymerbonding operation may include aligning the first substrate and thesecond substrate, mounting the first substrate and the second substrate,and baking the first substrate and the second substrate at a temperatureless than 100 degrees Celsius.

Another aspect of the present disclosure pertains to a method ofmanufacturing a flow cell. The method includes providing a firstsubstrate having a base substrate, a soft bottom layer on the basesubstrate, and a coated top layer, stamping a surface chemical patternby stamping through the coated top layer and stop beneath an interfaceof the soft bottom layer and the coated top layer, exposing the firstsubstrate to an adhesive promoter, exposing the first substrate to abio-medium, forming a patterned polymer layer on the first substrate ora second substrate, and polymer bonding the first substrate to thesecond substrate. The adhesive promoter reacts and attaches only to thecoated top layer and the bio-medium attaches to only the adhesivepromoter. The first substrate or the second substrate or both substratesare transparent. In some embodiments, the coated top layer is aspin-on-glass (SOG) oxide. In some embodiments, the soft bottom layer isa thermal plastic resin.

The present disclosure also pertains to a flow cell having a transparentsubstrate and a bottom substrate, which may or may not be transparent.One of the substrates has through holes. The flow cell also includes apatterned photoresist dry film between the first substrate and thesecond substrate. The patterned polymer layers form microfluidic channelwalls between the first substrate and the substrate. A plurality ofpatterned capture sites within microfluidic channels on the firstsubstrate, the second substrate, or both. The plurality of capture siteseach comprises a patterned adhesive promoter and a bio-medium. Thepatterned adhesive promoter and the bio-medium may be covalently bonded.The patterned capture sites may be disposed on a surface chemicalpattern having silicon oxide islands each disposed on an outcrop of asoft bottom layer over a base substrate. The soft bottom layer may bealuminum, copper, titanium, an alloy of these, or a combination of theseor a thermal plastic resin. The plurality of patterned capture sites maybe recessed into the first substrate, the second substrate, or both,formed directly on a planar surface of the first substrate, the secondsubstrate, or both. The first substrate and the second substrate may beabout or less than 1 mm thick and may be formed of quartz. Thebio-medium is a hydrogel.

In describing one or more of these embodiments, the present disclosuremay offer several advantages over prior art devices. In the discussionof the advantages or benefits that follows it should be noted that thesebenefits and/or results may be present is some embodiments, but are notrequired in every embodiment. Further, it is understood that differentembodiments disclosed herein offer different features and advantages,and that various changes, substitutions and alterations may be madewithout departing from the spirit and scope of the present disclosure.

What is claimed is:
 1. A flow cell comprising: a first substrate; asecond substrate; a first resin layer disposed over an inner surface ofthe first substrate; a second resin layer disposed over an inner surfaceof the second substrate; a first plurality of biological capture siteslocated at the first resin layer; a second plurality of biologicalcapture sites located at the second resin layer; and a polymer layerinterposed between the first resin layer and the second resin layer,such that the first substrate is attached to the second substrate via atleast the first resin layer, the polymer layer, and the second resinlayer, wherein the polymer layer defines a plurality of microfluidicchannels that extend through polymer layer.
 2. The flow cell of claim 1,comprising a plurality of recesses in which the first plurality ofbiological capture sites and the second plurality of biological capturesites are disposed.
 3. The flow cell of claim 1, wherein each of theplurality of biological capture sites comprises an adhesive promoter anda bio-medium.
 4. The flow cell of claim 4, wherein the bio-medium is ahydrogel.
 5. The flow cell of claim 3, wherein the adhesive promotor isa silane-based adhesive promotor.
 6. The flow cell of claim 3, whereinthe adhesive promotor comprises hexamethyldisilazane or 3-aminopropyltriethoxysilane.
 7. The flow cell of claim 1, wherein a thickness of thefirst substrate and a thickness of the second substrate areapproximately 1 mm or less.
 8. The flow cell of claim 1, wherein one orboth of the first substrate and the second substrate are transparent. 9.The flow cell of claim 8, wherein one or both of the first substrate andthe second substrate are made of glass.
 10. The flow cell of claim 1,wherein the first plurality of biological capture sites are offset fromthe second plurality of biological capture sites in a top view.
 11. Theflow cell of claim 1, wherein the first plurality of biological capturesites are aligned with the second plurality of biological capture sitesin a top view.
 12. The flow cell of claim 1, wherein a through holeextends through the first substrate and the first resin layer.
 13. Amethod of forming a flow cell, the method comprising: attaching a firstsubstrate that comprises a first plurality of recesses in each of whicha biological capture site is disposed to a second substrate via apolymer layer.
 14. The method of claim 13, comprising, before attachingthe first substrate to the second substrate, forming the biologicalcapture site in each of the recesses.
 15. The method of claim 14,wherein each capture site comprises an adhesive promoter and abio-medium.
 16. The method of claim 15, wherein forming the biologicalcapture site in each of the recesses comprises: depositing the adhesivepromoter at least in the first plurality of recesses of the firstsubstrate, and depositing the bio-medium on the adhesive promoter in thefirst plurality of recesses of the first substrate.
 17. The method ofclaim 15, wherein forming the biological capture site in each of therecesses comprises: depositing the adhesive promoter in the firstplurality of recesses of the first substrate and on portions of thefirst substrate around the first plurality of recesses, depositing thebio-medium on the adhesive promoter, and removing at least portions ofthe adhesive promoter that are not in the first plurality of recesses ofthe first substrate.
 18. The method of claim 17, wherein the step ofremoving at least portions of the adhesive promoter that are not in thefirst plurality of recesses of the first substrate is performed bychemical-mechanical polishing (CMP).
 19. The method of claim 15, whereinthe bio-medium is a hydrogel.
 20. The method of claim 15, wherein theadhesive promotor is a silane-based adhesive promotor.
 21. The method ofclaim 15, wherein the adhesive promotor comprises hexamethyldisilazaneor 3-aminopropyl triethoxysilane.
 22. The method of claim 13, whereinthe second substrate comprises a second plurality of recesses in each ofwhich a biological capture site is disposed.